This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ion channels are membrane proteins that play a central role in membrane permeability. Since passing molecules back and forth across the membrane is central to communication, it is not surprising that closing and opening a pore is the end result of a large class of ion channels. Channels have evolved specific domains to interact with their environment by binding ligands or interacting with the electric field across the membrane. These changes are allosterically coupled to the central pore of the channel causing it to open and allow ion flow. It is believed that the architecture of the conduction pore is common to all of these channels. Extensive mutational and structural data implicate the pore-lining inner helix as a gate that adopts different conformations when channels open and close. In the closed state, the channels pore-lining inner helices form an inverted 'teepee'resulting in a helical bundle at the apex that blocks the ion pathway. In the open state, these inner helices are thought to splay out, away from the central pore, allowing an unobstructed pathway for ions to move through the channel. In this project, we will focus on conformational changes of the pore-lining inner S6 helix of the voltage-gated potassium channel Kv1.2 and how single amino acid mutations affect these conformations. It is known that gating in voltage-dependent channels is very sensitive to a single amino acid mutations[1,2]. We will examine mutations that change the mechanics of the channel. Our motivation for this project is that this sensitivity of gating to mutational changes may provide valuable information about the mechanism of channel gating. We intend to look at the mechanical properties of a single alpha-helix that makes up pore lining using molecular dynamics (MD) simulations. We focus on residues preceding the Pro-Val-Pro (PVP) motif and their mutations because they are critical for helix bending and channel gating. Proline introduces helix flexibility by disrupting hydrogen bonding with the i-4 residue. This disruption of backbone hydrogen bonding and introduction of unusual dihedral angles often provides the basis for helix kinking. In addition to equilibrium simulations, we will also carry out umbrella sampling to compare free energy of bending for each mutation. Our initial analysis of the S6 helix from Kv1.2 revealed that the dihedral angles preceding the PVP motif are extremely far from ideal. During the equilibrium simulations, the extreme dihedral angles relax to typical alpha-helical values. This results suggest that the dihedral angles of pre-proline residues are good reaction coordinates for helix bending. Our preliminary result on free energy analysis seems consistent with recent experimental energy shifts. In order to confirm this consistency we must carry out more extensive MD simulations, especially for the umbrella sampling where we need two-dimensional reaction coordinates (e.g., two dihedral angles such as phi and psi) for free energy calculation of the helix bending. Our initial simulations focus on a single helix in explicit water. We will use NAMD for all MD simulations. The number of atoms in each system (one wild-type and five mutants) is about 32000. The estimated computing time based on initial test runs on a single Intel 2.4GHz CPU is as follows: 1200 SUs for initial setups (minimization, heating, and equilibration) and 28800 SUs for production runs (both equilibrium simulations and the umbrella sampling), i.e., total 250ns simulation for each system. After we carry out these simulations we intend to apply for a larger grant to extend our analyses to full channel structure in a membrane. References [1] O. Yifrach and R. MacKinnon, Cell, Vol 111, 231-239 (2002). [2] D.H. Hackos, T. Chang, and K.J. Swartz, J. Gen. Physiol, Vol. 119, 521-531 (2002).